The present invention relates to nanoparticles containing polymeric nucleic acid homologs, pharmaceutical compositions and articles of manufacture containing same and methods of use thereof and, more particularly, to delivery of functional polymeric nucleic acid homologs to living cells.
There is a long standing interest in development of new methods for gene delivery that do not require viral vectors which are typically associated with immunogenicity, oncogenesis and unknown long-term effects (Mahato et al. (1999) Adv Genet 41: 95-156). Development of these new methods has the potential to provide pharmaceutical formulations to deliver genes and to compete against conventional pharmaceutical, biological and surgical therapies, by offering increased efficacy, safety, regulatory compliance and reduced cost (Ledley (1994) Curr Opin Biotechnol 5: 626-636).
Unfortunately, known nonviral gene delivery systems or “artificial viruses” typically share two inherent disadvantages. The first disadvantage is that they are less effective than real viruses in terms of DNA delivery capacity. The second disadvantage is that they imitate biological functions of viruses and are therefore likely to cause an immune response and/or oncogenesis. Known nonviral gene delivery systems differ fundamentally from viral systems in terms of their composition, therapeutic profile, clinical risks and environmental safety (Ledley (1995) Hum Gene Ther 6: 1129-1144; Rolland (1998) Crit Rev Ther Drug Carrier Syst 15: 143-198 and Roy et al. (1999) Nature Med 5: 387-391). Cationic liposomes are one type of nonviral gene delivery systems (Chonn & Cullis (1998) AdvDrug Del Rev 30: 73-83. Cationic liposomes have as inherent disadvantages low in-vivo transfection efficiency and moderate cell toxicity. Further, cationic liposomes are subject to inactivation by serum and extracellular matrix components, and often form aggregates. These disadvantages render them unsuitable for use in many in-situ applications, although they are often employed in tissue culture.
Polymeric and collagen-based matrices are an additional type of nonviral gene delivery systems (Bonadio et al. (1998) Adv Drug Del Rev 33: 53-69. Labhasetwvar et al. (1998) JPharm Sci 1998; 87:1347-1350 and Shea et al (1999) Nat Biotechnol 17: 551-554). Matrices of these types are suitable only for local delivery. Further, they exhibit low in-vivo transfection efficiency, and large amounts of pDNA are required in order to achieve a biological effect. These disadvantages render them unsuitable for use in many in-situ applications, especially if a systemic effect is required or if local delivery is impractical.
Biodegradable and non-biodegradable polymer based particles are an additional type of nonviral gene delivery systems (Smith et al. (1997) Adv Drug Del Rev 26: 135-150; Mathiowitz et al. (1997) Nature 386: 410-414; Hedley et al. (1998) Nature Med 4: 365-368; Leong et al. (1998) J Control Rel 53: 183-193 and Labhasetwar et al. (1999) Colloids and Surfaces B:Biointerfaces 16: 281-290). While it is recognized that delivery via polymeric particles has the potential to offer increased resistance to nuclease degradation, control in dosing, sustained release of plasmid DNA (pDNA), and prolonged gene action (Ledley (1996) Pharm Res 13: 1595-1614 and Jong et al. (1997) J Control Rel 1997; 47: 123-134), this potential has not been realized in any specific clinical context to date.
Polymeric particle based systems are also theoretically able to control release rate kinetics, so as to provide a timed release of pDNA, and are therefore theoretically suitable in situations when long-term localized gene expression meets the aims of therapy. Unfortunately, development of sustained release gene delivery systems has been slow and fraught with technical difficulties (Labhasetwar et al. (1998) J Pharm Sci 87: 1347-1350; Shea et al. (1999) Nat Biotechnol 17: 551-554; Labhasetwar et al. (1999) Colloids and Surfaces B:Biointerfaces 16: 281-290; Jong et al. (1997) J Control Rel 47: 123-134; Luo et al. (1999) Pharm Res 16:1300-1308 and Ando et al. (1999) J Pharm Sci 88: 126130). Matrices of these types are suitable only for local delivery. Further, they exhibit low in-vivo transfection efficiency, and large amounts of pDNA are required in order to achieve a biological effect. These disadvantages render them unsuitable for use in many in-situ applications, especially if a systemic effect is required or if local delivery is impractical.
Thus, the primary obstacle preventing development of efficient non-viral gene delivery systems remains development of sufficient transfection efficiency. To date, direct transfection efficiency of pDNA from nanoparticles encapsulating pDNA (rather than transfection evaluation of) has not been reported. Instead, reported results have been in terms of transfection efficiency of pre-released pDNA. Similarly, absorbtion of pDNA containing particles and/or expression of pDNA encoded genes over prolonged periods of time has not been reported.
One proposed application for nanoparticle based gene delivery systems is antisense therapy. The purpose of antisense therapy is to inhibit expression of a particular gene. This may be achieved by using oligodeoxynucleotide (ODN), double stranded DNA peptide nucleic acids (PNA), antisense RNA, double stranded RNA or ribozymes. Antisense therapy works by inhibiting expression of a selected gene at the level of translation (Deshpande et al. (1996) Pharmaceutical News 3). For any protein or peptide to be synthesized, the gene encoding it must be transcribed from DNA to mRNA. In some cases the mRNA is processed, for example by splicing or cleavage. The mRNA must then be translated into protein. Antisense therapy relies upon down regulation of the functional protein by preventing mRNA translation or processing. This is typically achieved by destabilizing selected mRNA transcripts by hybridizing them with a complementary nucleic acid (or nucleic acid analog) sequence. The hyridized double stranded molecule is then subject to enzymatic degradation (e.g. with RNAase H) within the cell. Alternately, the hybridized double stranded molecule prevents mRNA processing because a recognition site for an enzyme is hidden by the complementary second strand. Alternately, the complementary second strand blocks the translation initiation codon of the transcript and prevents translational initiation (translational arrest). Phosphorothioated oligodeoxyribonucleotides (ODNs) stretches have been employed for sequence specific hybridization.
An alternate method of inhibiting a specific gene is transcriptional inhibition or the antigen approach. This may be achieved by use of PNAs (FIG. 1) which recognize and invade double-stranded DNA (triplex formation) in a sequence specific manner thereby preventing transcription. Alternately, PNA's may be employed as mRNA binding reagents in place of ODNs in antisense and antigen therapy as described above.
While the methods described hereinabove are theoretically recognized, their implementation has been problematic.
ODNs are usually prepared with a nuclease resistant derivatization such as phosphorothioate (PT-ODNs, FIG. 1) in order to retard enzymatic degradation and permit their activity in-vivo. Unfortunately, once in-vivo stability was achieved, even non-specific oligonucleotide sequences (PT-ODNs) have demonstrated unexpected potent activity (Stein (2001) J. Clin. Invest. 108:641-4). Furthermore, therapeutic use of ODNs has been hampered by their inherently low cellular permeability which is generally attributed to large molecular size and high charge density. The observed activity of ODNs in-vivo is therefore attributed to cellular uptake via an energy-dependent endocytosis (Akhtar et al. (1992) Trends Cell Biol. 2:139-144). These problems with ODNs have led to the development of viral vectors, liposomes and receptor-mediated endocytosis (Mahato et al. (1997) J. Drug Target. 4:337-357). Each of these delivery strategies has inherent disadvantages as detailed hereinabove and hereinbelow.
In summary, currently available viral gene delivery vectors share, as inherent disadvantages, a number of safety related problems including, but not limited to, immunogenicity of the vectors, formation of replication-competent viruses in patients and possible presence of contaminating agents in viral vector preparations. Additional safety concerns are raised by the possibility of insertional mutagenisis and transmission of viral and other exogenous DNA to the germ line of patients undergoing treatment. Additional disadvantages result from the physical limitations imposed by the virus particle. These additional disadvantages include lack of tissue specificity, limited insert size, and difficulties in manufacturing (Romano et al. (2000) Stem Cells. 18:19-39; Romano et al. (1999) Stem Cells. 17:191-202, Dani (1999) Braz. J. Med. Biol. Res. 32:133-145).
In summary, currently available non-viral gene delivery vectors share, as inherent disadvantages, low transfection efficiency, lack of specific targeting capability, transient expression and an immune response elicited from the unmethylated CpG islands of the bacterial DNA (Romano et al. (2000) Stem Cells. 18:19-39; Li et al. (2000). Gene Ther. 7:31-34; Rolland (1998) Crit. Rev. Ther. Drug Carrier Syst. 15:143-198). Liposomes have additional disadvantages as detailed herein above. Further, currently available non-viral gene delivery vectors rely upon receptor-mediated endocytosis which is largely due to poly-1-lysin as the gene vector. The use of poly-1-lysin is problematic due its polydispersity and heterogenicity which contribute to low in-vivo transfection efficiency.
While adsorbtion of ODNs onto polyalkylcyanoacrylate nanoparticles (Lambert et al. (2001) Adv. Drug. Del Rev. 47(1):99-112) enhances stability against nucleases and leads to a more ideal cellular distribution, problems of efficient cellular delivery, lysosomal degradation and lack of specificity have provided serious obstacles for therapeutic use of such particles.
Peptide nucleic acids (PNA) are ODN analogs in which the sugar-phosphate moiety has been replaced by 2-aminoethyl glycine units linked by amide bounds (FIG. 2 and Nielsen (2000) Curr. Opin. Mol. Ther. 2:282-7). The bases are attached to the backbone by methylene carbonyl linkage. Due to the backbone neutrality, PNA oligomers hybridize to complementary DNA and RNA faster and with greater affinity by Watson-Crick's base pairing (Ray & Norden (2000) FASEB J. 14:1041-60 and Soomets et al. (1999) Front. Biosci. 1:4D782-6). In addition, PNA are better at discriminating between base pair mismatches and have less affinity for cellular proteins than PT-ODNs (Doyle D F et al. (2001) Biochemistry. 40:53-64). Despite these advantages, PNAs are currently considered to be of only limited suitability for therapeutic use because of their poor cellular incorporation (Dean (2000) Adv. Drug Del. Rev. 44:81-95).
One medical problem which may be amenable to antisense therapy is restenosis after percutaneous transluminal coronary angioplasty. Restenosis is the main complication after percutaneous transluminal coronary angioplasty and accounts for 35 to 40% of post procedure complications (Chorny et al. (2000) Crit. Rev. Ther. Drug. Carrier. Syst. 17:249-84). The migration and proliferation of medial vascular smooth muscle cells (SMC) are thought to be the main components in neointimal formation. Signal transduction through the platelet-derived growth factor (PDGF)/PDGF receptors system is involved in the process of post-angioplasty restenosis. A sustained inhibition of this pathway is therefore a promising strategy which has gained considerable research attention as a potential means to effectively control neointimal formation (Fishbein et al. (2000) Arterioscler Thromb Vasc Biol. 20:667-76; Fishbein et al. (2000) J. Controlled Release. 65:221-9; Hughes et al. (1996) Gen. Pharmac. 27:1079-1089; Noiseux et al. (2000) Circ. 102:1330-1336 and Sirois et al. (1997) Circulation. 95:669-676). However, no significant progress has been made in exploiting the PDGF/PDGF receptors system as a point of intervention for prevention of restenosis. This lack of progress results from an emphasis on drug based, ae opposed to gene based, therapy. Gene therapy has the potential to be far mor specific by use of, for example, promoter specific sequences or cell specific ligands.
Antisense therapy also has potential utility in treatment of cell proliferation disorders. For example, altering intimal hyperplasia has been reported using AS-ODNs to inhibit growth-regulatory or cell-cycle genes (c-myb, c-myc, PCNA, cdc2, and cdk2) involved in SMC proliferation Sirois et al. (1997) Circulation. 95:669-676. and Villa et al. (1995) Circ. Res. 76:505-513. However, all the AS-ODNs used were fully phosphorothioated analogs. Although fully phosphorothioated ODNs have increased stability and nuclease resistance, they display high affinity for various cellular proteins, resulting in nonspecific effects. Furthermore, high concentrations of phosphorothioated oligodeoxynucleotides (PT-ODNs) inhibit DNA polymerases and RNase H, which may render them ineffective as antisense agents (Stein (2001) J. Clin. Invest. 108:641-4).
The most common problems encountered in prior art gene therapy protocols are poor efficacy and immune response of the host to the vector. Poor efficacy may result from failure of the delivered material to enter cells, to integrate into the genome, or to be expressed at appropriate levels. In addition, response over the course of time is often poor. This means that readministration, which might be advantageous, is often problematic due to the abovementioned immune response.
There is thus a widely recognized need for, and it would be highly advantageous to have, nanoparticles containing polymeric nucleic acid homologs, pharmaceutical compositions and articles of manufacture containing same and methods of use thereof devoid of the above limitations.